Shear stress and microRNAs for better metastatic cancer management.

Metastasis is the process by which cancer cells move from the primary location to establish themselves in a new location in the human body. It is still a significant challenge in cancer management because it is responsible for 90% of cancer-related deaths. In this work, we present an idea to use shear stress encountered by all metastasizing cells as an elegant means to deactivate metastasizing cancer cells. Shear-induced ROS and cross-talk between ROS and miRNA play crucial roles in deactivating metastasizing cancer cells. In addition, there exists a vast therapeutic potential for miRNAs. Therefore, this study explores the effect of shear on miRNAs and reactive oxygen species (ROS), the two molecular mediators in the proposed {shear-stress}-{miRNA}-{metastasizing-cancer-cell-deactivation} approach. In this context, to understand the effect of defined shear on HCT116 colon cancer cells, they were cultivated in a defined shear environment provided by an appropriately designed and fabricated cone-and-plate device. Shear rate affected the culture growth characteristics and the specific intracellular reactive oxygen species level (si-ROS). HCT116 cell growth was observed at 0 and 0.63 s-1 but not at 1.57 s-1 or beyond. Shear rate induced upregulation of the hsa-miR-335-5p but induced downregulation of hsa-miR-34a-5p. Furthermore, the specific levels of hsa-miR-335-5p, hsa-miR-26b-5p, and hsa-miR-34a-5p negatively correlated with specific intracellular (si)-hydroxyl radical levels. In addition, some messenger RNAs (mRNAs) in HCT116 cells showed a differential expression under shear stress, notably the ROS-associated mRNA of PMAIP1. The above miRNAs (and possibly some mRNAs) could be targeted to manage colon cancer metastasis.

Improved redox anti-cancer treatment efficacy through reactive species rhythm manipulation.

Rhythms in the pseudo-steady state (PSS) levels of reactive species (RS), particularly superoxide and hydroxyl radicals, exist in cancer cells. The RS rhythm characteristics, particularly frequency and amplitude, are entrained (reset) by the anticancer compounds/drugs. In this work, we show for the first time that the phase of the RS rhythm at which the drug is added is significantly important in determining the cytotoxicity of anticancer compounds/drugs such as menadione and curcumin, in two different cancer cell lines. Curcumin, the more effective of the two drugs (IC50 = 15 µM, SiHa; 6 µM, HCT116) induced reset of superoxide and hydroxyl rhythms from 15.4 h to 9 h, and 25 h to 11 h respectively, as well as caused increases in these radical levels. However, menadione (IC50 = 20 µM, SiHa; 17 µM, HCT116) affected only the superoxide levels. Drug treatment at different time points/phase of the RS rhythm resulted in a maximum of 27% increase in cytotoxicity, which is significant. Further, we report for the first time, an unexpected absence of a correlation between the intracellular PSS RS and antioxidant levels; thus, the practice of using antioxidant enzyme levels as surrogate markers of intracellular oxidative stress levels may need a re-consideration. Therefore, the RS rhythm could be a fundamental/generic target to manipulate for improved cancer therapy.

Discrimination of driver and passenger mutations in epidermal growth factor receptor in cancer.

Cancer is one of the most life-threatening diseases and mutations in several genes are the vital cause in tumorigenesis. Protein kinases play essential roles in cancer progression and specifically, epidermal growth factor receptor (EGFR) is an important target for cancer therapy. In this work, we have developed a method to classify single amino acid polymorphisms (SAPs) in EGFR into disease-causing (driver) and neutral (passenger) mutations using both sequence and structure based features of the mutation site by machine learning approaches. We compiled a set of 222 features and selected a set of 21 properties utilizing feature selection methods, for maximizing the prediction performance. In a set of 540 mutants, we obtained an overall classification accuracy of 67.8% with 10 fold cross validation using support vector machines. Further, the mutations have been grouped into four sets based on secondary structure and accessible surface area, which enhanced the overall classification accuracy to 80.2%, 81.9%, 77.9% and 75.1% for helix, strand, coil-buried and coil-exposed mutants, respectively. The method was tested with a blind dataset of 60 mutations, which showed an average accuracy of 85.4%. These accuracy levels are superior to other methods available in the literature for EGFR mutants, with an increase of more than 30%. Moreover, we have screened all possible single amino acid polymorphisms (SAPs) in EGFR and suggested the probable driver and passenger mutations, which would help in the development of mutation specific drugs for cancer treatment.

Intrinsic apoptosis and NF-κB signaling are potential molecular targets for chemoprevention by black tea polyphenols in HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo.

Antiproliferative and apoptosis inducing effects of black tea polyphenols (Polyphenon-B) on HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo were investigated. Viability of HepG2 cells was evaluated by the MTT assay, and apoptosis by AO-EB and DAPI staining, cell cycle analysis, and annexin V-PI assay. For the in vivo study, male Sprague-Dawley rats treated with dimethylaminoazobenzene (DAB) (0.06%) were used. The expression of Bcl-2 and NF-κB family members were analyzed by immunoblotting. Administration of Polyphenon-B induced dose-dependent inhibition of growth of HepG2 cells and reduced tumor incidence in DAB administered animals. HepG2 cells also exhibited morphological features characteristic of apoptotic cell death. In addition, administration of Polyphenon-B increased the expression of Bax, tBid, Smac/Diablo, cytochrome C, Apaf-1, caspases, and IκB with PARP cleavage, and decreased the expression of Bcl-2, Bcl-xL, pBad, NF-κB, p-IκB-α, IKKß and Ub in both HepG2 cells and in DAB-treated animals. These results provide evidence that Polyphenon-B effectively inhibits proliferation and induces apoptosis both in vitro and in vivo by inhibiting NF-κB, and inducing intrinsic apoptosis by modulating the expression of a network of interrelated molecules eventually culminating in caspase-mediated cell death.

The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-κB inhibition.

With increasing use of plant-derived cancer chemotherapeutic agents, exploring the antiproliferative effects of phytochemicals has gained increasing momentum for anticancer drug design. The dietary phytochemical quercetin, modulates several signal transduction pathways associated with cell proliferation and apoptosis. The present study was undertaken to examine the effect of quercetin on cell viability, and to determine the molecular mechanism of quercetin-induced cell death by investigating the expression of Bcl-2 family proteins (Bcl-2, Bcl-xL, Mcl1, Bax, Bad, p-Bad), cytochrome C, Apaf-1, caspases, and survivin as well as the cell cycle regulatory proteins (p53, p21, cyclin D1), and NF-κB family members (p50, p65, IκB, p-IκB-α, IKKß and ubiquitin ligase) in human cervical cancer (HeLa) cells. The results demonstrate that quercetin suppressed the viability of HeLa cells in a dose-dependent manner by inducing G2/M phase cell cycle arrest and mitochondrial apoptosis through a p53-dependent mechanism. This involved characteristic changes in nuclear morphology, phosphatidylserine externalization, mitochondrial membrane depolarization, modulation of cell cycle regulatory proteins and NF-κB family members, upregulation of proapoptotic Bcl-2 family proteins, cytochrome C, Apaf-1 and caspases, and downregulation of antiapoptotic Bcl-2 proteins and survivin. Quercetin that exerts opposing effects on different signaling networks to inhibit cancer progression is a classic candidate for anticancer drug design.

The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells.

Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-kappaB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.

Anti-inflammatory effect of curcumin involves downregulation of MMP-9 in blood mononuclear cells.

Curcumin (1, 7-bis (4-hydroxyl-3-methoxyphenyl)-1, 6 heptadiene-3, 5-dione) is a potent natural anti oxidant and anti-inflammatory agent, which mediates its effects mainly by inhibiting the activity of enzymes like cyclooxygenase, lipooxygenases and phospholipase A2. Here we examined the possibility of curcumin affecting the production of matrix metalloproteinases (MMPs) by peripheral blood mononuclear cells (PBMCs), which play an important role in inflammation. Zymographic analysis and ELISA showed that curcumin significantly inhibited the activity and level of MMPs produced by PBMCs isolated from human and inflammation-induced rabbit in a concentration dependent manner. The administration of curcumin to inflammation-induced rabbits also caused downregulation of MMP-9. Kinetic analysis showed that the effect of curcumin was a delayed one indicating inhibition of de novo protein synthesis. RT-PCR and immunoblot analysis showed inhibition of the production of MMP-9 mRNA and protein respectively by human PBMCs, which were activated in vitro by Artocarpus Lakoocha agglutinin (ALA) lectin. EMSA and super shift showed activation of classical NFkappaB in in vitro activated PBMCs and treatment with curcumin inhibited activation of NFkappaB. Immunoblot analysis suggested that ALA-induced activation of NFkappaB leading to the upregulation of MMP-9 was due to the degradation of IkappaB-alpha. Curcumin inhibited the degradation of IkappaB-alpha, which inhibited the ALA mediated activation of NFkappaB and upregulation of MMP-9. These results indicated that anti-inflammatory effect of curcumin also involves inhibition of the production of MMP-9 in PBMCs.

Induction of apoptosis by curcumin and its implications for cancer therapy.

Curcumin (diferuloyl methane), the yellow pigment in turmeric (Curcuma longa), is a potent chemopreventive agent that inhibits proliferation of cancer cells by arresting them at various phases of the cell cycle depending upon the cell type. Curcumin-induced apoptosis mainly involves the mitochondria-mediated pathway in various cancer cells of different tissues of origin. In some cell types like thymocytes, curcumin induces apoptosis-like changes whereas in many other normal and primary cells curcumin is either inactive or inhibits proliferation, but does not appear to induce apoptosis. These together with reports that curcumin protects cells against apoptosis induced by other agents, underscore the need for further understanding of the multiple mechanisms of cell death unleashed by curcumin. Tumor cells often evade apoptosis by expressing several antiapoptotic proteins, down-regulation and mutation of proapoptotic genes and alterations in signaling pathways that give them survival advantage and thereby allow them to resist therapy-induced apoptosis. Many researchers including ourselves, have demonstrated the involvement of several pro and antiapoptotic molecules in curcumin-induced apoptosis, and ways to sensitize chemoresistant cancer cells to curcumin treatment. This review describes the mechanisms of curcumin-induced apoptosis currently known, and suggests several potential strategies that include down-regulation of antiapoptotic proteins by antisense oligonucleotides, use of proapoptotic peptides and combination therapy, and other novel approaches against chemoresistant tumors. Several factors including pharmacological safety, scope for improvement of structure and function of curcumin and its ability to attack multiple targets are in favor of curcumin being developed as a drug for prevention and therapy of various cancers.

Changes in matrix metalloproteinases and their endogenous inhibitors during tumor progression in the uterine cervix.

PURPOSE: To study the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cervical tumorigenesis, we analyzed 70 cervical tissue specimens that included 15 low-grade squamous intraepithelial lesions (SILs), 20 high-grade SILs, 25 squamous cell carcinomas (SCCs) and 10 specimens of normal cervical tissue. METHODS: The gelatinolytic activity of MMP-9 and MMP-2 was determined by zymographic analysis. The expression of MMP-9 and MMP-2 and TIMP-1 and TIMP-2 was determined by immunohistochemistry. RESULTS: All the samples had 72/66 kDa gelatinase activity; 92 kDa gelatinase activity was detected only in high-grade SILs and SCCs. Immunohistochemical analysis showed weak positivity for MMP-2 in normal cervical epithelium and low-grade SILs. However, high-grade SILs and SCCs showed intense cellular and stromal reactivity for MMP-2 and MMP-9. For TIMP-1 and TIMP-2, normal cervical epithelium and low-grade SILs showed intense immunostaining, >50% of high-grade SILs showed positivity, and 95% of SCCs showed intense stromal and cellular reactivity. CONCLUSIONS: Increase in the relative activity of these gelatinases and enhanced immunostaining for MMPs and TIMPs with tumor progression suggest that they may play a crucial role in cervical cancer progression. A significant association between stage of the lesion and expression of MMPs and TIMPs ( P<0.01) was found. Immunohistochemical studies indicate that these MMPs may be of basal cell origin in cervical tissue, although the mechanism of their upregulation is not clearly understood.

Human colon cancer cells differ in their sensitivity to curcumin-induced apoptosis and heat shock protects them by inhibiting the release of apoptosis-inducing factor and caspases.

Mild heat treatment induced the expression of heat shock protein-70 (hsp70), hsp90 and hsp27 in two human colon cancer cell lines, one derived from primary tumor, SW480, and the other derived from the secondary lymph node tissue, SW620, of the same patient. SW620 cells appear to be more sensitive to curcumin-induced apoptosis. Heat shock protects both the human colon cancer cells from curcumin-induced apoptosis. Heat shock prevented, at least in part, the release of apoptosis inducing factor from mitochondria induced by curcumin although the release of second mitochondria derived activator of caspase and cytochrome c was unaffected in both the cells. Moreover, heat shock reduced curcumin-induced activation of caspases 9 and 3 but not 8.

Cytotoxic activity of amooranin and its derivatives.

Amooranin, 25-hydroxy-3-oxoolean-12-en-28-oic acid, is a triterpene acid isolated from Amoora rohituka stem bark. The cytotoxic effects of amooranin and its derivatives were studied. Amooranin and its methyl ester showed greater cytotoxicity against MCF-7 and HeLa cells derived from tumour tissues with a 50% inhibitory concentration (IC(50)) of 1.8-3.4 microg/mL, compared with Chang liver cells from normal tissue with an IC(50) of 6.2-6.4 microg/mL, but amooranin exhibited no activity on HEp-2 and L-929 cells. However, its monoacetate derivative showed no inhibitory activity at 1-10 microg/mL dose levels. Of the cytotoxic isolates, the methyl ester derivative was inactive in in vivo evaluations in the Ehrlich ascites tumour cells at 50 and 100 mg/kg/day, demonstrating T/C values of 106% and 114%, respectively.

L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis.

Curcumin (diferuloyl methane), the yellow pigment in turmeric (Curcuma longa), is a potent chemopreventive agent. Curcumin induces apoptosis of several, but not all, cancer cells. Many cancer cells protect themselves against apoptosis by activating nuclear factor-kappaB (NF-kappaB)/Rel, a transcription factor that helps in cell survival. Signal-induced activation of NF-kappaB is known to be inhibited by curcumin. To understand the role of NF-kappaB in curcumin-induced apoptosis, we stably transfected relA gene encoding the p65/RelA subunit of NF-kappaB, into l-929 cells (mouse fibrosarcoma) and the relA-transfected cells were resistant to varying doses of curcumin (10(-6)-10(-4) m), whereas the parental cells underwent apoptosis in a time- and dose-dependent manner. The relA-transfected cells showed constitutive NF-kappaB DNA binding activity that could not be inhibited by curcumin and did not show nuclear condensation and DNA fragmentation upon treatment with curcumin. When a super-repressor form of IkappaB-alpha (known to inhibit NF-kappaB) was transfected transiently into relA-transfected cells, the cells were no longer resistant to curcumin. Our results highlight a critical anti-apoptotic role for NF-kappaB in curcumin-induced apoptosis.

Involvement of cdc2-mediated phosphorylation in the cell cycle-dependent regulation of p185neu.

We previously reported cell cycle-dependent negative regulation of p185neu (decreased tyrosine phosphorylation and kinase activity, with electrophoretic mobility retarded by serine/threonine phosphorylation) in M phase and the escape of mutation-activated p185neu* from this regulation. Our present results showed that retardation of electrophoretic mobility occurs independently of the cells' transformed status. We found that normal p185neu lost its ability to dimerize in the M phase. We demonstrated a physical association between cdc2 (a serine/threonine kinase, active in M phase) and p185neu. We showed that the carboxy terminal portion of p185neu is phosphorylated in vitro by cdc2. Many phosphopeptides (at least three phosphoserine residues) unique to the M phase were identified, and the in vivo and in vitro phosphopeptide patterns were superimposable. In contrast, mutation-activated p185neu* dimerized in the M phase with no changes in electrophoretic mobility, failed to associate with cdc2 and no unique phosphoserine residues could be identified in the M phase (data not shown), consistent with the escape of p185neu* from cell cycle-dependent regulation. Our results suggest that this escape is an intrinsic property of the mutation-activated p185neu* independent of its ability to transform cells. Our results also suggest the involvement of serine/threonine kinases such as cdc2 in the cell cycle-dependent negative regulation of p185neu.

Mitosis-specific negative regulation of epidermal growth factor receptor, triggered by a decrease in ligand binding and dimerization, can be overcome by overexpression of receptor.

The function of epidermal growth factor receptor (EGFR) was found to be negatively regulated in M phase in which it showed less phosphotyrosine content and reduced intrinsic kinase activity accompanied by retarded electrophoretic mobility owing to total hyperphosphorylation. Ligand-induced autophosphorylation and downstream signaling of EGFR were tightly suppressed in M phase due to a decrease in ligand binding affinity and the inability of epidermal growth factor (EGF) to induce receptor dimerization. There was no change in the number of surface-exposed EGF receptors between G0/G1 and M phases of the cell cycle. Hyperphosphorylation (due to serine and/or threonine phosphorylation) correlates with the unresponsiveness of cells to EGF-mediated stimulation of tyrosine phosphorylation in cells that express the normal or basal level of EGFR. This M phase-specific negative regulation was overcome by overexpression of EGFR, which was responsive to ligand throughout the cell cycle and revealed ligand-induced signaling in the M phase. These findings indicate that EGFR does not respond to ligand stimulation in M phase and suggest that a negative regulation of ligand-receptor interactions in M phase may control the normal function of receptor tyrosine kinase and that receptor overexpression will disrupt this cell cycle-dependent regulation of receptor tyrosine kinases.

Mapping of adenovirus 5 E1A domains responsible for suppression of neu-mediated transformation via transcriptional repression of neu.

Overexpression of neu (also known as c-erbB-2 or HER-2) commonly occurs in human cancer and is also known to enchance tumor metastasis and chemoresistance. Our earlier reports showed that the adenovirus 5 E1A can suppresss the neu-mediated transformation by repression of neu. Thus, E1A has the potential to be used as a therapeutic agent against the neu-overexpressing human cancers. However, a serious concern to this approach is that E1A is also capable of immortalizing primary culture cells and can co-operate with ras or E1B oncogenes to transform them. The E1A CR2 domain (amino acid residues 120 to 140) necessary for binding to RB is believed to be required for this oncogenic function. Here, we report that deletion of CR2 region did not affect E1A's capability to repress neu. Interestingly, deletion of the amino acid residues 4 to 25 or 40 to 80 completely disrupted E1A-mediated neu repression. By deleting the amino acid residues from 81 to 185, we have successfully generated a mini-E1A mutant that was sufficient to inhibit neu promoter activity and suppress neu-mediated transformation. The mini-E1A mutant does not contain the CR2 domain that is crucial for RB binding and immortalization, and hence, may serve as a more selective tumor suppressor, and a safer therapeutic agent. It may also be a useful tool to further investigate the molecular mechanism(s) of neu overexpression and E1A-mediated transcriptional repression in cancer cells.

Inhibition of nuclear factor-kappaB activity is involved in E1A-mediated sensitization of radiation-induced apoptosis.

The adenoviral E1A protein has been implicated in the potentiation of apoptosis induced by various external stimuli, but the exact mechanism of that potentiation is not clear. In this study, we compared the sensitivity to ionizing gamma-irradiation of E1A transfectants with that of parental cells in a human ovarian cancer cell line (SKOV3.ip1); we found that the E1A transfectants became sensitive to radiation-induced apoptosis. Recently, activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to play a key role in the anti-apoptotic pathway of radiation-induced apoptosis. In an attempt to determine whether NF-kappaB was involved in the E1A-mediated sensitization of radiation-induced apoptosis, we found that radiation-induced activation of NF-kappaB occurred in the parental cells but was blocked in the E1A transfectants. Furthermore, parental cells cotransfected with NF-kappaB and E1A were better protected from undergoing apoptosis upon irradiation than those transfected with E1A alone. Thus, our results suggest that inhibition of NF-kappaB activation by E1A is a plausible mechanism for E1A-mediated sensitization of radiation-induced apoptosis.

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.

An immunological approach reveals biological differences between the two NDF/heregulin receptors, ErbB-3 and ErbB-4.

The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB-2), and two receptors for the Neu differentiation factor (NDF/heregulin), namely: ErbB-3 and ErbB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB-3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB-3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase-defective ErbB-3, probably with ErbB-2, is essential for transmission of NDF signals through ErbB-3.

ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.

Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

ErbB-3 mediates differential mitogenic effects of NDF/heregulin isoforms on mouse keratinocytes.

The family of Neu differentiation factors (NDFs, or heregulins) includes a dozen secreted glycoproteins, whose receptor binding domain displays two variants, alpha and beta, and they bind to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Certain isoforms were reported to induce growth-arrest and differentiation of mammary tumor cells, while other breast cancer cell lines responded mitogenically. The present study addressed the biologic effects of various NDF isoforms on normal EGF-dependent epithelial cells, Balb/MK keratinocytes, that can undergo either proliferation or differentiation. We found that beta isoforms of NDF induced a mitogenic effect, that was significantly smaller than the maximal response to EGF. By contrast with NDF-beta, NDF-alpha isoforms exerted almost no mitogenic effect, but they were sufficient to maintain keratinocytes in culture. Consistent with their higher mitogenic potency, NDF-beta isoforms bound to Balb/MK cells with higher affinity (Kd = 2.2 nM) than alpha isoforms, however both groups shared their receptor, that we identified as ErbB-3. No transcript of ErbB-4 was detectable in the keratinocytes, but these cells express multiple NDF mRNAs and also ErbB-2. We conclude that different isoforms of NDF induce distinct growth regulatory effects on cultured keratinocytes, through direct interaction with ErbB-3.